Visual imaging of molecules and/or processes ongoing in living tissue is an area of scientific and medical importance which, despite scientific advances, is still underdeveloped. Visualization of soft tissue is of particular value to the medical imaging industry and to the pharmaceutical industry. In medical imaging, there is a constant need for imaging agents (contrast agents or diagnostic agents) that enhance the assessment of one or more of healthy tissue, a disease process affecting tissue, and a disease state of affected tissue. Typically, medical imaging involves delivery of an imaging agent to an organ or tissue to be imaged. Generally, an organ or tissue is imaged is to determine the presence or absence of a suspected abnormality. As to the pharmaceutical industry, in drug development it is particularly important to monitor one or more of: (a) the distribution of the drug in a particular target organ or tissue; (b) the interaction of the drug with living cells of the organ or tissue; (c) internalization of the drug by tissue cells, when the target of action is intracellular; and (d) metabolism or bioclearance of the drug in living tissues.
Typically, conventional fluorescent labels (e.g., fluorescein, rhodamine, phycoerythrin, an the like) are used to study biochemical, pharmacological, or pathological changes that occur in tissue by first fixing the tissue. However, such fluorescent labels are not suitable for all biological applications. For example, conventional fluorescent labels are generally toxic to living cells and tissues comprised of living cells. Additionally, conventional fluorescent labels generally suffer from short-lived fluorescence; e.g., undergo photobleaching after minutes of exposure to an excitation light source. Thus, they would not be suitable for visual imaging requiring any significant length of time needed to ascertain the complexities of a biological process. Further, conventional fluorescent labels are sensitive to changes in environment which can decrease their quantum yield; e.g., brought about by changes in the surrounding pH and dissolved oxygen. Changes in the surrounding pH and dissolved oxygen are conditions that typically may be encountered in living tissues (including organs). Another disadvantage of conventional fluorescent labels is that typically the excitation spectrum of a species of fluorescent label may be quite narrow. However, even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different species of fluorescent labels to permit individual and quantitative detection without substantial spectral overlap. Thus, when using a combination of different fluorescent labels, multiple filters are typically needed to detect the resultant emission spectra of the combination. Conventional fluorescent labels are limited in sensitivity and resolution of imaging due to the limitations of intensity, photobleaching, and the finite number of molecules which can be used to label a substrate.
Semiconductor nanocrystals ("quantum dots") are known in the art. Generally, quantum dots can be prepared which result in relative monodispersity; e.g., the diameter of the core varying approximately less than 10% between quantum dots in the preparation. Examples of quantum dots are known in the art to have a core selected from the group consisting of CdSe, CdS, and CdTe (collectively referred to as "CdX"). CdX quantum dots have been passivated with an inorganic coating ("shell") uniformly deposited thereon. Passivating the surface of the core quantum dot can result in an increase in the quantum yield of the fluorescence emission, depending on the nature of the inorganic coating. The shell which is used to passivate the quantum dot is preferably comprised of YZ wherein Y is Cd or Zn, and Z is S, or Se. Quantum dots having a CdX core and a YZ shell have only been soluble in organic, non-polar (or weakly polar) solvents. Thus, the instability of these quantum dots in aqueous media has limited their usefulness in biological applications.
To make quantum dots useful in biological applications, it is desirable that the quantum dots are water-soluble. "Water-soluble" is used herein to mean sufficiently soluble or suspendable in a aqueous-based solution, such as in water or water-based solutions or physiological solutions, including those used in biological or molecular detection systems as known by those skilled in the art. Typically, CdX core/YZ shell quantum dots are over-coated with trialkylphosphine oxide, with the alkyl groups most commonly used being butyl and octyl. One method to make the CdX core/YZ shell quantum dots water-soluble is to exchange this overcoating layer with a coating which will make the quantum dots water-soluble. For example, a mercaptocarboxylic acid may be used to exchange with the trialkylphosphine oxide coat. Exchange of the coating group is accomplished by treating the water-insoluble quantum dots with a large excess of neat mercaptocarboxylic acid. Alternatively, exchange of the coating group is accomplished by treating the water-insoluble quantum dots with a large excess of mercaptocarboxylic acid in CHCl.sub.3 solution (Chan and Nie, 1998, Science 281:2016-2018). The thiol group of the new coating molecule forms Cd (or Zn)--S bonds, creating a coating which is not easily displaced in solution. Another method to make the CdX core/YZ shell quantum dots water-soluble is by the formation of a coating of silica around the dots (Bruchez, Jr. et al., 1998, Science 281:2013-2015). An extensively polymerized polysilane shell imparts water solubility to nanocrystalline materials, as well as allowing further chemical modifications of the silica surface. However, depending on the nature of the coating group, quantum dots which have been reported as water-soluble may have limited stability in an aqueous solution, particularly when exposed to air (oxygen) and/or light. More particularly, oxygen and light can cause the molecules comprising the coating to become oxidized, thereby forming disulfides which destabilize the attachment of the coating molecules to the shell. Thus, oxidation may cause the coating molecules to migrate away from the surface of the nanocrystals, thereby exposing the surface of the nanocrystals in resulting in "destabilized nanocrystals". Destabilized nanocrystals form aggregates when they interact together, and the formation of such aggregates eventually leads to irreversible flocculation of the nanocrystals.
Thus, current fluorescent molecules (fluorescent labels and quantum dots) are not suitable for labeling of live tissues and visual imaging. In that regard, provided herein are fluorescent labels that are: (a) functionalized to enhance stability under the complex conditions of aqueous environments encountered in living tissues (including organs); (b) stable in the varying conditions encountered in labeling protocols for living tissue; (c) non-toxic to living tissue; (d) extremely sensitive in terms of detection, because of their fluorescent properties (e.g., including, but not limited to, high quantum efficiency, resistance to photobleaching, and stability in complex aqueous environments); (e) a class of semiconductor nanocrystals that may be excited with a single wavelength of light resulting in detectable fluorescence emissions of high quantum yield and with discrete fluorescence peaks; and (f) functionalized so as to be bound to an affinity ligand which is used to target the tissue to be imaged by fluorescence.